A break in both strands of the DNA double helix is one of the most harmful DNA lesions because if not properly repaired it leads to mutations, chromosome rearrangements or cell death. The safest way to repair a DNA double-strand break (DSB) is via homologous recombination (HR). The known mechanisms of HR utilize an intact homologous DNA sequence as a template for DNA polymerase to retrieve the information lost at the site of a DSB and to generate an overhang required for re-joining of the broken DNA ends. Here, we hypothesized that RNA can serve as a template for DNA DSB repair either directly or indirectly in the form of a complementary DNA, cDNA, via HR mechanisms. Our previous work in vivo in yeast and in vitro demonstrated a new role of RNA in DNA repair. We showed that both synthetic RNA oligonucleotides and endogenous transcript RNA can support repair of DSBs in chromosomal DNA in yeast via HR either directly or after conversion into cDNA. We also found that deficiency of ribonuclease H function stimulates DNA repair by RNA. Considering the abundance of RNA in cells, the flow of genetic information from RNA to DNA could markedly impact genome stability. However, very little is known about the mechanisms that activate and regulate transfer of genetic information from RNA to DNA during DSB repair by HR. The experiments proposed in this grant application will characterize the mechanisms of these RNA-mediated events. We will utilize budding yeast and human model systems, and by employing genetic, molecular and cellular biology and biochemical assays we will analyze the molecular processes how both cDNA and transcript RNA direct DSB repair via HR. We will focus on the following Aims: 1) Identify key proteins and critical steps of DSB repair via HR directed by transcript RNA in yeast. A combination of reverse and forward genetic approaches will be applied to uncover genetic pathways of RNA-mediated DNA break repair via HR. 2) Reconstitute the mechanism of RNA-directed DSB repair in vitro using yeast and human proteins. In vitro reconstitution systems will be then used to identify yeast and human factors that promote DSB repair directed by RNA-templates. 3) Examine whether the process of RNA-mediated DSB repair via HR is conserved in mammalian cells. Assays to study DSB repair by RNA will be developed. Transfection experiments and flow cytometry will be used to determine whether RNA transcript and/or cDNA can provide homologous templates for DSB repair in mammalian dividing and non-dividing cells.